qiime2安装和使用案例

 QIIME是微生物组领域最广泛使用的分析流程。本文章介绍conda 安装Linux（64-bit）以及使用脚本。数据为illumina PE250的fq文件。

1. 安装

conda update conda
conda install wget
wget https://data.qiime2.org/distro/core/qiime2-2021.8-py38-linux-conda.yml
conda env create -n qiime2-2021.8 --file qiime2-2021.8-py38-linux-conda.yml
# OPTIONAL CLEANUP
rm qiime2-2021.8-py38-linux-conda.yml

2. 输入数据到QIIME 2 artifact

qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path manifest --output-path demux-paired-end.qza --input-format PairedEndFastqManifestPhred33

qiime demux summarize --i-data demux-paired-end.qza --o-visualization demux-paired-end.qzv

3. 去噪和OTU聚类

qiime dada2 denoise-paired \
  --i-demultiplexed-seqs demux-paired-end.qza \
  --p-trunc-len-f 250 \
  --p-trunc-len-r 250 \
  --p-n-threads ${threads} \
  --o-table dada2_table.qza \
  --o-representative-sequences dada2_rep-seqs.qza \
  --o-denoising-stats dada2_stats.qza

qiime metadata tabulate \
  --m-input-file dada2_stats.qza  \
  --o-visualization dada2_stats.qzv

qiime feature-table summarize \
  --i-table dada2_table.qza \
  --o-visualization dada2_table.qzv

#导出代表序列
qiime tools export --input-path dada2_rep-seqs.qza --output-path phyloseq

#导出特征表
qiime tools export --input-path dada2_table.qza --output-path exported-feature-table
# 格式转化
biom convert -i exported-feature-table/feature-table.biom -o exported-feature-table/feature-table.txt --to-tsv

4. OTU物种注释

qiime feature-classifier classify-sklearn \
  --p-n-jobs ${threads} \
  --i-classifier ${silva_classifier_qza} \
  --i-reads dada2_rep-seqs.qza \
  --o-classification taxonomy.qza                                                  
qiime metadata tabulate \
  --m-input-file taxonomy.qza \
  --o-visualization taxonomy.qzv

qiime taxa barplot \
  --i-table dada2_table.qza \
  --i-taxonomy taxonomy.qza \
  --o-visualization taxa-bar.qzv

#  导出OTU注释表
qiime tools export --input-path taxonomy.qza --output-path taxonomy

# 导出个样本微生物丰度文件
qiime tools export \
  --input-path taxa-bar.qzv \
  --output-path bar_taxonomy

5. 比对代表性序列，并构建系统发育树

qiime phylogeny align-to-tree-mafft-fasttree \
  --p-n-threads ${threads} \
  --i-sequences dada2_rep-seqs.qza \
  --o-alignment aligned-rep-seqs.qza \
  --o-masked-alignment masked-aligned-rep-seqs.qza \
  --o-tree unrooted-tree.qza \
  --o-rooted-tree rooted-tree.qza

#导出进化树
qiime tools export --input-path unrooted-tree.qza --output-path exported-tree

6. 阿尔法稀释作图

qiime diversity alpha-rarefaction \
  --i-table dada2_table.qza \
  --i-phylogeny rooted-tree.qza \
  --p-max-depth 4000 \
  --o-visualization alpha-rarefaction.qzv

更多应用请参考  https://docs.qiime2.org/2021.8/